Neurofibromin, a neurofibromatosis type I (NF1) tumor suppressor gene product, has a domain acting as a GTPase activating protein and functions in part as a negative regulator of Ras. Loss of neurofibromin expression in NF1 patients is associated with elevated Ras activity and increased cell proliferation. Therefore, regulation of the function of neurofibromin is heavily involved in cell growth and differentiation. In the present study, we identified a novel cellular neurofibromin‐associating protein, 14‐3‐3, which belongs to a highly conserved family of proteins that regulate intracellular signal transduction events in all eukaryotic cells. The interaction of 14‐3‐3 is mainly directed to the C‐terminal domain (CTD) of neurofibromin, and the cAMP‐dependent protein kinase (PKA)‐dependent phosphorylation clustered on CTD‐Ser (2576, 2578, 2580, 2813) and Thr (2556) is required for the interaction. Interestingly, the increased phosphorylation and association of 14‐3‐3 negatively regulate the function of neurofibromin. These findings indicate that PKA phosphorylation followed by 14‐3‐3 protein interaction may modulate the biochemical and biological functions of neurofibromin.